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Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Pirotte N, Stevens AS, Fraguas S, Plusquin M, Van Roten A, Van Belleghem F, Paesen R, Ameloot M, Cebrià F, Artois T, Smeets K - Oxid Med Cell Longev (2015)

Bottom Line: Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis.ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process.Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium.

ABSTRACT
Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

No MeSH data available.


Related in: MedlinePlus

DPI exposure does not affect stem cell proliferation. (a) Cell proliferation in DPI-exposed (2 μM) head, trunk, and tail fragments at 4, 48, and 72 hours post amputation. No significant effects of DPI exposure on cell proliferation were observed (n ≥ 3). (b) DPI exposure did not affect stem cell proliferation in intact animals. 7 days of DPI exposure before immunostaining (n ≥ 5). (c) Schematic figure of the cell cycle and visualization of the different stem cell populations. (d) FACS data of the three stem cell populations (X1, X2, and Xins) during DPI exposure in comparison with control animals at 4 HPA and 72 HPA. Each sample at 4 HPA existed of 6 regenerating head fragments, while measurements at 72 HPA were performed with 4 animals/sample. No effects of the DPI exposure were observed on any of the different neoblast populations or on their distributions.
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fig5: DPI exposure does not affect stem cell proliferation. (a) Cell proliferation in DPI-exposed (2 μM) head, trunk, and tail fragments at 4, 48, and 72 hours post amputation. No significant effects of DPI exposure on cell proliferation were observed (n ≥ 3). (b) DPI exposure did not affect stem cell proliferation in intact animals. 7 days of DPI exposure before immunostaining (n ≥ 5). (c) Schematic figure of the cell cycle and visualization of the different stem cell populations. (d) FACS data of the three stem cell populations (X1, X2, and Xins) during DPI exposure in comparison with control animals at 4 HPA and 72 HPA. Each sample at 4 HPA existed of 6 regenerating head fragments, while measurements at 72 HPA were performed with 4 animals/sample. No effects of the DPI exposure were observed on any of the different neoblast populations or on their distributions.

Mentions: Stem cells (neoblasts) are the underlying force of successful regeneration [17, 19, 24, 25]. Despite drastic regeneration defects caused by a diminished ROS production, DPI did not affect the regular stem cell proliferation peaks at 4 hours and 72 hours post amputation. Neither the number of cells in the G2/M phase at 4 HPA, 48 HPA, or 72 HPA (Figure 5(a)), nor the different neoblast populations (X1: proliferating neoblasts in G2/M phase; X2: nonproliferating neoblasts in G0/G1 phase; Xins: differentiating cells) were altered during DPI exposure in regenerating fragments (Figures 5(c) and 5(d)) [61]. Gene expression analyses of the neoblast-specific proliferation marker pcna and cell cycle regulating genes cdc73 and cyclinB-1 confirm the lack of effects on stem cell proliferation (Figure 3, supplementary Table 1). However, gene expression of tor (target of rapamycin) was downregulated during DPI exposure in regenerating head fragments at 4 HPA (p = 0.032) (Figure 3, supplementary Table 1). TOR is a widely conserved and important regulator of cell growth and proliferation, controlling the progression of the G1 phase to the S phase of the cell cycle [62–64]. Mammalian studies have reported that ROS (H2O2) activate TOR and its target S6 kinase [65]. Moreover, TOR is not only activated by increased ROS levels, TOR itself also regulates the production of ROS [65]. Literature shows that, similar to DPI-exposed phenotypes, tor(RNAi) fragments are unable to form recognizable blastemas and regenerate the nervous system structures (e.g., brain, visual neurons) within preexisting tissues which do not require the formation of these structures [62–64]. Intact tor(RNAi) treated animals develop lesions, comparable to the APO phenotype, but these lesions were present in the postpharyngeal region instead of the head region [62, 64]. This suggests that the cell cycle might not be completely unaffected by an impaired ROS production, as was also shown by the decrease in proliferation which was observed in regenerating tails and fins of Xenopus and zebrafish, respectively, after ROS reduction [6, 7]. On the other hand, we must keep in mind that proliferative responses during regeneration in Xenopus species and zebrafish were measured on mitotic, dedifferentiated epidermal cells, while regeneration in S. mediterranea is the result of proliferation of pluripotent stem cells.


Reactive Oxygen Species in Planarian Regeneration: An Upstream Necessity for Correct Patterning and Brain Formation.

Pirotte N, Stevens AS, Fraguas S, Plusquin M, Van Roten A, Van Belleghem F, Paesen R, Ameloot M, Cebrià F, Artois T, Smeets K - Oxid Med Cell Longev (2015)

DPI exposure does not affect stem cell proliferation. (a) Cell proliferation in DPI-exposed (2 μM) head, trunk, and tail fragments at 4, 48, and 72 hours post amputation. No significant effects of DPI exposure on cell proliferation were observed (n ≥ 3). (b) DPI exposure did not affect stem cell proliferation in intact animals. 7 days of DPI exposure before immunostaining (n ≥ 5). (c) Schematic figure of the cell cycle and visualization of the different stem cell populations. (d) FACS data of the three stem cell populations (X1, X2, and Xins) during DPI exposure in comparison with control animals at 4 HPA and 72 HPA. Each sample at 4 HPA existed of 6 regenerating head fragments, while measurements at 72 HPA were performed with 4 animals/sample. No effects of the DPI exposure were observed on any of the different neoblast populations or on their distributions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: DPI exposure does not affect stem cell proliferation. (a) Cell proliferation in DPI-exposed (2 μM) head, trunk, and tail fragments at 4, 48, and 72 hours post amputation. No significant effects of DPI exposure on cell proliferation were observed (n ≥ 3). (b) DPI exposure did not affect stem cell proliferation in intact animals. 7 days of DPI exposure before immunostaining (n ≥ 5). (c) Schematic figure of the cell cycle and visualization of the different stem cell populations. (d) FACS data of the three stem cell populations (X1, X2, and Xins) during DPI exposure in comparison with control animals at 4 HPA and 72 HPA. Each sample at 4 HPA existed of 6 regenerating head fragments, while measurements at 72 HPA were performed with 4 animals/sample. No effects of the DPI exposure were observed on any of the different neoblast populations or on their distributions.
Mentions: Stem cells (neoblasts) are the underlying force of successful regeneration [17, 19, 24, 25]. Despite drastic regeneration defects caused by a diminished ROS production, DPI did not affect the regular stem cell proliferation peaks at 4 hours and 72 hours post amputation. Neither the number of cells in the G2/M phase at 4 HPA, 48 HPA, or 72 HPA (Figure 5(a)), nor the different neoblast populations (X1: proliferating neoblasts in G2/M phase; X2: nonproliferating neoblasts in G0/G1 phase; Xins: differentiating cells) were altered during DPI exposure in regenerating fragments (Figures 5(c) and 5(d)) [61]. Gene expression analyses of the neoblast-specific proliferation marker pcna and cell cycle regulating genes cdc73 and cyclinB-1 confirm the lack of effects on stem cell proliferation (Figure 3, supplementary Table 1). However, gene expression of tor (target of rapamycin) was downregulated during DPI exposure in regenerating head fragments at 4 HPA (p = 0.032) (Figure 3, supplementary Table 1). TOR is a widely conserved and important regulator of cell growth and proliferation, controlling the progression of the G1 phase to the S phase of the cell cycle [62–64]. Mammalian studies have reported that ROS (H2O2) activate TOR and its target S6 kinase [65]. Moreover, TOR is not only activated by increased ROS levels, TOR itself also regulates the production of ROS [65]. Literature shows that, similar to DPI-exposed phenotypes, tor(RNAi) fragments are unable to form recognizable blastemas and regenerate the nervous system structures (e.g., brain, visual neurons) within preexisting tissues which do not require the formation of these structures [62–64]. Intact tor(RNAi) treated animals develop lesions, comparable to the APO phenotype, but these lesions were present in the postpharyngeal region instead of the head region [62, 64]. This suggests that the cell cycle might not be completely unaffected by an impaired ROS production, as was also shown by the decrease in proliferation which was observed in regenerating tails and fins of Xenopus and zebrafish, respectively, after ROS reduction [6, 7]. On the other hand, we must keep in mind that proliferative responses during regeneration in Xenopus species and zebrafish were measured on mitotic, dedifferentiated epidermal cells, while regeneration in S. mediterranea is the result of proliferation of pluripotent stem cells.

Bottom Line: Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis.ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process.Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

View Article: PubMed Central - PubMed

Affiliation: Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, 3590 Diepenbeek, Belgium.

ABSTRACT
Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes.

No MeSH data available.


Related in: MedlinePlus